This project involves the study of the relationships between nitrogen metabolism and that of fatty acids and ketoacids in the following in vitro systems: (1) primary cultures of astrocytes; (2) primary cultures of neurons; and (3) cultured skin fibroblasts taken from patients with inherited disorders of fatty acid oxidation, particularly medium chain and long chain acyl-CoA dehydrogenase deficiencies. The core hypothesis with regard to the work in the brain cells is that excessive levels of fatty acids impair normative metabolism of glutamine, glutamate and aspartate and that the encephalopathy characteristic of patients with defective lipid oxidation of inherited or acquired (e.g., Reye's syndrome) etiology is in part referable to excessive levels of such neurotoxic compounds as glutamate and aspartate. The core hypothesis underlying the research involving cultured skin fibroblasts is that the hyperammonemia in patients with acquired or primary defects of fatty acid oxidation is secondary not only to impaired hepatic ureagenesis, but also to excessive ammonia production brought about by a shift in the glutamate dehydrogenase reaction toward the oxidative deamination of glutamate rather than the reductive amination of alpha-ketoglutarate. This shift occurs because the failure of lipid oxidation leads to a relative depletion of intra-mitochondrial acetyl-CoA and a consequent reduction in the intra-mitochondrial concentration of alpha- ketoglutarate. We have identified a similar, though not identical, adaptation to explain the augmentation of renal ammoniagenesis observed in metabolic acidosis. These hypotheses will be tested by incubating highly purified cultures of astrocytes or neurons as well as cultured skin fibroblasts from dehydrogenase-deficient patients with 15N labelled precursors, e.g., (2-15N)glutamine, and utilizing gas chromatography-mass spectrometry to quantitate isotopic abundance in such products as (15N)aspartate, (15N)glutamate, 15NH3, (15N)glutamic acid, etc. Our prior work has shown the stable isotope 15N to be an extremely useful metabolic tracer for the description of the nitrogen metabolism in in vitro systems. By utilizing gas chromatography-mass spectrometry for the quantitation of isotopic enrichment in the indicated products, precursor-product relationships and net rates of nitrogen flux can be determined with a rigor which would otherwise not be possible.